TIE2-expressing monocytes regulate revascularisation of the ischaemic limb

BackgroundMonocytes (CD14+ cells) expressing the receptor TIE2 are a highly angiogenic subset that are pivotal to neovascularisation in the tumour environment. We hypothesised that TIE2-expressing monocytes (TEMs) are also important in neovascularisation of ischaemic tissues.MethodsFlow cytometry was used to quantify circulating TEMs in 40 patients with critical limb ischaemia (20 age-matched and 20 healthy controls). RT-PCR was used to confirm TIE2 expression in FACS-sorted TEMs. ELISA was used to measure circulating levels of the TIE2 ligand angiopoietin 2 (ANG2). Mice were subjected to hindlimb ischaemia and TEMs quantified. In an additional study, haemopoietic stem/progenitor cells, isolated from Pgk-rtTA-miR-126T transgenic mice, were transduced ex vivo with a TRE-miR-Tie2-OFP lentiviral vector and used to reconstitute lethally irradiated mice. These mice were treated with alternate daily doses of doxycycline to silence Tie2 expression on TEMs, and hindlimb ischaemia (HLI) was induced. Conversely, bone-marrow-derived macrophages (BMDMs) were enforced to express Tie2 using a Pgk-Tie2 lentivirus and delivered into the ischaemic hindlimb. Recovery of ischaemia was measured with laser Doppler.FindingsFlow cytometry revealed a ten-fold higher number of circulating TEMs in patients with critical limb ischaemia than in matched controls (mean 3·52% [SE 0·28] vs 0·39 [0·09], p<0·0001). Revascularisation or amputation resulted in a fall in TEM numbers to control levels (p<0·005). Analysis by RT-PCR confirmed TIE2 mRNA expression in TEMs. Circulating ANG2 levels were two-fold higher in patients with critical limb ischaemia than in controls (mean 4354 pg/mL [SE 661] vs 1973 [247], p<0·05). Circulating CD115+/Tie2+ monocyte numbers were higher following hindlimb ischaemia in mice than in sham controls (p<0·05) and Tie2 expression was upregulated in CD45+/CD11b+/F4/80+ macrophages isolated from ischaemic hindlimbs (p<0·05). Tie2 gene knockdown in TEMs inhibited neovascularisation of the ischaemic hindlimb (p<0·0001). Delivery of Tie2-expressing BMDMs into the ischaemic limb accelerated the recovery of blood flow compared with treatment with control BMDMs.InterpretationOur studies suggest that TEMs are mobilised following ischaemia and contribute to the revascularisation of ischaemic tissue, and that TIE2 is important for the proangiogenic functios of TEMs. TEMs may represent a promising, novel therapeutic target for cell therapy in critically ischaemic tissues.